The vision of point-of-care pathogen detection has become more achievable since the development of isothermal nucleic acid amplification techniques. These techniques rapidly produce large quantities of nucleic acids to enable high sensitivity of detection, whilst only requiring a single temperature of operation. Recombinase polymerase amplification (RPA) is a relatively new isothermal amplification technique amenable for use in low-resource settings. Detection can be achieved using lateral flow devices, components are supplied freeze-dried in single use ampoules, and the assay operates at 37 °C. However, little is known about the tolerance of RPA for the detection of heterogeneous sequence populations. Indeed, the extended length requirements for RPA primers and probe can make it more difficult to find near-exact regions of conservation within diverse sequence populations.
Here we describe an RPA assay for detection of Japanese encephalitis virus (JEV), which comprises 4 lineages and 7 clusters, with approximately 20% sequence variability between divergent isolates. The virus is of global public health concern, and remains the most common cause of viral encephalitis in Asia, despite the implementation of a vaccine. A rapid field-based assay would help identify endemic regions and monitor virus spread. We performed a multiple sequence alignment of 162 JEV sequences in the NS5 gene, and identified regions suitable for assay development. Resultant primers and probes contained 20 - 30% degeneracy, and were iteratively tested to select an optimized set. Despite the high level of degeneracy, the resultant assay was selective for JEV when tested against a panel of highly related flaviviruses, including Kunjin, Kokobera, Murray Valley encephalitis, and Alfuy viruses. The results indicate that high sequence specificity may be an added advantage of the RPA assay. Further methodical testing of RPA sequence tolerance would be beneficial for determining the stringency of the assay for mismatch incorporation.